Investigating Environmental Determinants of Soil-Transmitted Helminths Transmission using GPS Tracking and Metagenomics Technologies

Background: The Global Health community aims to eliminate soil-transmitted helminth (STH) infections by 2030. Current preventive methods such as Mass Drug Administration, WASH practices, and health education needs to be complimented to halt transmission. We tracked the movement of hookworm-infected and non-infected persons and investigated soil factors in the places they frequented within an endemic community to further understand the role of human movement and sources of infections. Methods: 59 positive and negative participants wore GPS tracking devices for 10 consecutive days and their movement data captured in real time. The data was overlaid on the community map to determine where each group differentially spent most of their time. Soil samples were collected from these identified sites and other communal places. Physical and chemical properties were determined for each sample using standard methods and helminth eggs cultured into larvae using the Baermann technique. Bivariate and multivariate analyses were used to determine associations between larvae counts and soil factors. Helminth species were identified with metagenomic sequencing and their distributions mapped to sampling sites in the community. Results: The study found that there was no significant difference in the average larvae counts in soil between sites assessed by infected and non-infected participants (P=0.59). However, soil factors, such as pH, carbon and sandy-loamy texture were associated with high larvae counts (P<0.001) while nitrogen and clay content were associated with low counts(P<0.001). The dominant helminth species identified were Panagrolaimus superbus (an anhydrobiotic helminth), Parastrongyloides trichosuri (a parasite of small mammals), Trichuris trichuria (whipworm), and Ancylostoma caninum (dog hookworm). Notably, no Necator americanus was identified in any soil sample. Conclusion: This study provides important insights into the association between soil factors and soil-transmitted helminths. These findings contribute to our understanding of STH epidemiology and support evidence-based decision-making for elimination strategies.


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Approximately 500 million individuals residing in tropical regions of Africa, South America, and 71 Asia are infected with hookworms, predominantly Necator americanus and Ancylostoma 72 duodenale [1]. These infections result in four million disability-adjusted life years (DALYs) [2] 73 and incur an estimated cost of USD 139 billion in annual economic productivity losses [3] The 74 primary clinical symptoms of hookworm disease include iron deficiency anaemia due to blood 75 loss, abdominal pains, diarrhoea, and protein malnutrition [4]. In children and women of 76 childbearing age, chronic infections and associated blood loss lead to low iron stores, which 77 impairs physical and cognitive development in children and increases perinatal maternal/infant 78 mortalities in pregnant women [5]. 79 To combat hookworm infections, various treatment and control strategies have been implemented, 80 including Mass Drug Administration (MDA), sanitation improvements and health education 81 programs[6]. These strategies have been effective in reducing the prevalence of hookworm 82 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint infection, but there is still a need for further research and implementation to completely eradicate 83 this disease. The high rates of reinfection following drug treatment and emerging drug resistance 84 pose major challenges to current deworming programs [7]. Hookworm infections are mainly 85 treated with single doses of either albendazole (400 mg) or mebendazole (500 mg)[4], [8] termed 86 preventive chemotherapy (PC) and is based on MDA to all at-risk individuals in endemic areas[9], 87 [10]. As of 2016, 638.5 million people had been covered, which included 69.5% of at-risk school-88 aged children and 50.8% of at-risk pre-school-aged children globally [11], [12]. These efforts were 89 further supplemented with the implementation of the Water, Sanitation and Hygiene (WASH) 90 programs to address the lack of potable water and sanitation facilities in schools [13]. 91 The situation in Ghana is comparable to other countries where parasitic infections, specifically 92 hookworms, are prevalent, with reported rates as high as 50% [11], [14], [15]. Our cohort studies 93 conducted in rural communities in the Kintampo North Municipality (KNM), Ghana 118 We, therefore, employed GPS technology to monitor the movements of hookworm-infected and 119 non-infected participants to identify the sites in the community that were likely sources of 120 infections and the soil properties that are associated with the presence of helminth larvae.

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Study area 123 Kawampe, the study site is a rural community in the Kintampo North Municipality (KNM), which 124 is located between latitudes 8º45'N and 7º45'N and longitudes 1º20'W and 2°1'E and, covers an       The trimmed reads for each sample were run against a database with assigned taxonomy 207 information using Kraken2 [49], which is a taxonomic classification system and database that uses 208 exact k-mer matches to achieve high accuracy and fast classification speeds. There are no available 209 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint standard Kraken2 taxonomy databases built for helminths, therefore, a customized database named 210 NematodeDB was built from the genome sequences of about 160 flatworm and nematode species.

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The taxonomic information of the genomes was obtained from the NCBI reference sequences

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Permission was obtained from the chief, elders, and opinion leaders of the study community.

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Informed consent was obtained from adults and assent/parental consent in the case of children.  CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The study participants 240 Out of the 59 participants who were monitored, 54.2% (n=32) were males, and 45.8% (n=27) were 241 females. The average age was 25.9 years (SD=±18.6; Range=6-83), and the majority (33.9%; 242 n=20) were aged between 11 and 20 years. Hookworm was present in 52% of the participants.   The Lima soil type was found mainly to the north of Kawampe at 10 sampled sites and, the rest 258 of the sites were Kumayili (Figure 2).

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. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint The participants' daily movements were mainly within the community and to neighbouring 265 farmlands ( Figure 3). These movements were mostly in the mornings and evenings and virtually 266 non-existent at night. The movement dynamics of both positive and negative adult participants 267 indicated a close interaction among community members (Figures 3A and 3B). Their daily 268 movements became more individualized due to the distinct ownership of farms ( Figure 3B). The 269 movements of children primarily revolved around the community, as their activities were centred 270 on community and school-based engagements.

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. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint which had a pH of 6.84 and a loamy sand texture.

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Soil factors that produced high larvae counts included soil pH, sandy-loamy soil type, and effective 286 cation exchange capacity (Figure 4). An increase in soil pH level was associated with an incidence 287 rate ratio of 3.7, (CI; 2.1-6.6, P< 0.001) based on the larvae counts and soil parameters from the 288 NBRM. Sandy-loamy soil had approximately 7 times more larvae compared to loamy soil, with 289 an incidence rate ratio of 6.7, (2.3-19.7, P<0.001) and Effective cation exchange capacity had an 290 incidence rate ratio of 1.3, (1.2-1.43, P<0.001).

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Interestingly, there were no hits for Necator americanus (human hookworm).  . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The global health community is targeting 2030 as the year to achieve the elimination of STH.

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Thus, any additional intervention tool that could support the existing MDA and WASH strategies 315 is a prerequisite for this to be achieved. Our studies, therefore, focused on tracking hookworm-316 infected individuals and gathering soil characteristics to identify specific areas in a community 317 that may contribute significantly to hookworm transmission [54]. Based on our study, the pH level 318 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint of soil was found to be a critical determinant of parasite growth, as it was linked to elevated larval  In this study, the soil type that exhibited the least conducive environment for larval density was 331 clay. Clay soil has limited air circulation and pore spaces, making it unsuitable for larvae 332 habitation. Soils that were primarily clay-based were found to have lower larvae counts [58]. Other CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint particularly rodents, which can be hosts for parasites commonly found around rubbish dumps. P. 349 trichosuri may have been introduced into the soil from such rodents. P. trichosuri is an interesting 350 nematode that can undergo multiple reproductive cycles as a free-living worm, enabling it to 351 increase the number of its infective L3s. Thus, it can be maintained in cultured conditions for 352 several free-living life cycles without requiring a permissive host, usually its marsupial host.

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Trichuris trichiura, also known as the human whipworm, was the third most common soil-

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. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint Lastly, the parasites may be localized to specific locations within the community which suggests 379 that a more elaborate sampling needs to be done to increase the chances of finding them [69], [70]. 380 This study on soil factors for soil-transmitted helminths (STH) has provided valuable insights into 381 the relationship between soil characteristics and the presence of some STH larvae. Tracking the 382 movements of participants has also shown significant convergence of people, whether infected or 383 not in particular places within the community. Sanitation and proper waste management in such 384 concentrated areas may reduce soil infestation.     WHO, "Helminth control in school-age children," 2011. 407 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 23, 2023. ; https://doi.org/10.1101/2023.07.17.23292808 doi: medRxiv preprint